Microbiology Lab Report: Bacterial Identification and Analysis

Introduction

The identification of bacteria is a meticulous and systematic process involving various techniques, including Gram staining, microscopic analysis, inoculation on selective and differential media, catalase testing, oxidase testing, and Microbact testing. These methods are employed to narrow down the types of bacteria present in an unknown bacterial culture.

In this case, a group of children at a childcare center experienced illness characterized by symptoms such as abdominal pain, nausea, vomiting, and diarrhea. All of the affected children had consumed peanut butter sandwiches for lunch the day before.

The childcare center had used Brand X peanut butter. Common pathogens responsible for gastrointestinal infections include Salmonella, Shigella, Campylobacter, invasive E. coli, and Yersinia.

The primary objective of this experiment is to identify all the bacteria present in our sample and determine whether these bacteria are pathogenic or part of the normal flora. Identifying pathogens is crucial for selecting appropriate antibiotics targeted at the identified pathogens.

Protocol

Week 1

1. Inoculation of Bacteria: Bacteria were inoculated onto nutrient agar plates using the 16-streak technique to promote bacterial reproduction and the formation of pure colonies for each bacterium present in the sample.

2. Incubation: Incubation was carried out at the appropriate temperature. For the peanut butter sample, incubation was performed at 37°C, while for Dam Water and Dish Cloth samples, the incubation temperature was maintained at 28°C.

Week 2

1. Subculturing of Bacteria: Bacteria were subcultured onto nutrient agar plates using the 16-streak technique. Blood Agar was used for the cultivation of fastidious bacteria with small colonies.

2. Oxidase Test: An oxidase test was conducted to determine the presence of cytochrome oxidase in the bacterial isolates.

3. Catalase Test: A catalase test was performed to detect the presence of catalase enzyme in the bacterial isolates.

4. Gram Stain: Bacteria were subjected to the Gram staining procedure to classify them as either Gram-positive or Gram-negative.

5. Microscopic Observation: Bacterial morphology was observed under the microscope at magnifications of 400X and 1000X.

6. Incubation: Incubation was carried out at the appropriate temperature, with the peanut butter sample incubated at 37°C, and the Dam Water and Dish Cloth samples at 28°C.

7. Record Keeping: Results were recorded on Laboratory Report Form 1.

Week 3

1. Inoculation of Pure Culture: Bacteria from pure cultures were inoculated onto selective and differential agar plates, including Blood Agar, MacConkey Agar, Mannitol Salt Agar, and Brilliance UTI Agar, using the 16-streak technique.

2. Microbact Test: Microbact tests were performed for Gram-negative bacteria that were also oxidase-negative.

3. Antibiotic Resistance Test: The antibiotic resistance of the bacterial isolates was determined using the E-disk diffusion technique.

4. Incubation: Incubation was conducted at the appropriate temperatures, with the peanut butter sample incubated at 37°C and the Dam Water and Dish Cloth samples at 28°C.

Week 4

1. Completion of Microbact Test: Reacting agents were added to the microbact tests post-incubation to complete the tests.

2. Observation of Bacterial Characteristics: The characteristics of the bacteria grown on each of the selective and differential agar plates were observed.

3. Measurement of Inhibition Zones: The inhibition zones on the Mueller-Hinton Agar (MHA) plates were measured to determine antibiotic resistance.

4. Record Keeping: Results were recorded in the laboratory report table provided by the course instructor.

Week 5

1. Identification of Bacteria: The bacteria present in the samples were identified based on the results obtained from the various tests and observations.

Results

The results of the tests have been recorded in three separate laboratory report forms. The first report provides information about the characteristics of the bacteria cells under examination, the second report pertains to the biochemical analysis (Microbact) conducted exclusively on Gram-negative and oxidase-negative bacteria, and the third report presents information regarding antimicrobial agents (antibiotics) and their effectiveness against the tested bacteria.

Bacteria Characteristics (Laboratory Report 1)

Characteristics	Peanut Butter Big Colony	Peanut Butter Fastidious
Cellular Morphology	Gram Negative	Gram Positive
Cellular Morphology	Bacillus	Cocci
Cell Arrangement	Single	Strepto
Colony Morphology	Gross Morphology	Gross Morphology
Colony Height	Flat	Flat
Colony Margins	Smooth	Smooth
Colony Size	Medium	Small
Colony Pigmentation	Opaque	Opaque
Colony Characteristic on Selective and Differential Media	Blood Agar: γ-haemolytic MacConkey: Non-lactose fermenting Bacteria Mannitol Salt Agar: No growth Brilliance Chromogenic UTI Agar: White	Blood Agar: β-haemolytic MacConkey: No growth Mannitol Salt Agar: No growth Brilliance Chromogenic UTI Agar: No growth
Biochemistry	Catalase Test: Positive Oxidase Test: Negative	Catalase Test: Negative Oxidase Test: Negative
Identification	Salmonella spp.	Streptococcus pyogenes

Antimicrobial Agents (Laboratory Report 3)

Antibiotic	Peanut Butter Big Colony	Peanut Butter Fastidious
Ampicillin	23 mm	35 mm
Gentamicin	20 mm	23 mm
Tetracycline	20 mm	30 mm
Chloramphenicol	25 mm	28 mm
Cephalothin	24 mm	36 mm
Clindamycin	R	26 mm

Discussion

Each of the procedures and techniques employed in the identification of the bacterial sample aims to provide valuable information for further characterization.

Gram Stain Procedure

The Gram stain procedure is a fundamental technique that classifies bacteria into two groups, Gram-positive and Gram-negative, based on their ability to retain the crystal violet-iodine complex. Gram-negative bacteria appear pink due to safranin staining, while Gram-positive bacteria appear deep violet. This staining pattern is determined by the structure and composition of the bacterial cell wall. In our case, we conducted two Gram stains, which revealed Gram-negative characteristics for the Peanut Butter Big Colony and Gram-positive characteristics for the Peanut Butter Fastidious bacteria.

Selective and Differential Media

Selective and differential media are essential tools in microbiology for isolating and identifying specific organisms. Selective media encourage the growth of certain organisms while inhibiting others, whereas differential media help differentiate closely related organisms or groups.

In our study, we inoculated two different bacteria onto four different plates containing various selective and differential media, yielding the following results:

Colony Characteristic on Selective and Differential Media	Peanut Butter Big Colony	Peanut Butter Fastidious
Blood Agar	γ-haemolytic	β-haemolytic
MacConkey	Non-lactose fermenting Bacteria	No growth
Mannitol Salt Agar	No growth	No growth
Brilliance Chromogenic UTI Agar	White	No growth

These results provide important insights into the characteristics of the bacteria under investigation:

• Blood Agar: The γ-haemolytic response indicates partial breakdown of red blood cells, leaving a greenish coloration, while β-haemolysis suggests complete lysis of red blood cells, resulting in a clear zone. The absence of hemolysis (γ-haemolysis) indicates no blood cell breakdown.
• MacConkey Agar: This selective and differential medium is useful for isolating non-fastidious gram-negative rods, particularly those belonging to the Enterobacteriaceae family. In our case, the non-lactose fermenting characteristics observed may be indicative of the presence of Salmonella or Escherichia coli.
• Mannitol Salt Agar (MSA): The lack of growth on MSA suggests that the high salt concentration in the medium inhibits the growth of most bacteria, except staphylococci. Staphylococcus aureus, a potential pathogen, can produce small colonies surrounded by yellow zones on MSA due to its ability to ferment mannitol and produce acid.
• Brilliance Chromogenic UTI Agar: This medium utilizes chromogenic substrates to differentiate bacteria based on enzymatic activities. Pink colonies indicate the presence of E. coli, while blue or turquoise colonies are indicative of enterococci. Dull blue or purple colonies suggest the presence of coliforms. This medium aids in the specific identification of these bacteria.

The oxidation test is used to determine the presence or absence of cytochrome oxidase in bacteria. Typically, this enzyme is present in aerobic bacteria. A positive reaction results in a purple or blue color on the strip, while a negative reaction shows no color change. In our oxidation test, we obtained negative reactions for all tested bacteria, which led us to consider the possibility of anaerobic or facultative anaerobic bacteria.

The catalase test assesses a bacterium's ability to metabolize hydrogen peroxide. In our tests, we observed a positive reaction for all types of bacteria, indicating that they are facultative anaerobic bacteria.

The Microbact test, our final step before identification, is a diagnostic testing system that aids in identifying organisms based on biochemical reactions. This test was conducted exclusively for Gram-negative and oxidase-negative bacteria, and the results were compared to a computerized probability list of known organisms.

The antibiotic susceptibility test involves the placement of antibiotic-impregnated discs on agar plates with bacterial cultures. After incubation, zones of growth inhibition around each antibiotic disc are measured to determine susceptibility (S), intermediate susceptibility (I), or resistance (R). These results allowed us to identify two different bacteria types in the sample: Streptococcus pyogenes and Salmonella species.

Salmonella Infection

Salmonella infection, also known as salmonellosis, is a common bacterial disease affecting the intestinal tract. Salmonella bacteria are typically found in the digestive tracts of animals and humans and can be transmitted through contaminated water or food. Symptoms may include diarrhea, fever, and abdominal cramps. While most healthy individuals recover without specific treatment, severe dehydration may require medical attention. In our case, the symptoms observed in the affected children after consuming peanut butter sandwiches are consistent with a Salmonella infection. Furthermore, the bacterial identification tests align with the presence of Salmonella in the peanut butter sample.

Streptococcus Pyogenes Infection

Infection with Streptococcus pyogenes, a beta-hemolytic bacterium belonging to Lancefield sero-group A, can cause a range of diseases in humans. S. pyogenes is a common cause of strep throat and impetigo, as well as more severe conditions like streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF). While S. pyogenes is commonly found in the normal flora, it can also cause gastrointestinal infections in humans. Treatment options may include antibiotics such as ampicillin or cephalothin, which have shown the highest effectiveness against this bacterium.

Conclusion

Based on the results obtained from various laboratory tests and analyses, we can conclude that the bacteria responsible for the illness among the children in the kindergarten is Salmonella spp. As discussed earlier, Salmonella causes gastrointestinal diseases. We recommend a treatment regimen that includes the antibiotic Chloramphenicol, as it demonstrated the highest effectiveness based on our results.

Additionally, we identified another bacterium in the sample, Streptococcus pyogenes. While this bacterium is commonly found in the normal flora, it can also be responsible for gastrointestinal infections in humans. Treatment options for S. pyogenes infections may include ampicillin or cephalothin antibiotics, as these demonstrated the best effectiveness against the bacterium in our tests.

It's worth noting that the Microbact test did not yield conclusive results in our study. This may have been due to various factors, including insufficient bacterial samples, reagent issues, or misinterpretation of color changes. Therefore, further optimization and validation of this test may be necessary for future studies.

References

1. Rai, S., Yadav, U., Pant, N., Yakha, J., Tripathi, P., Poudel, A., & Lekhak, B. (2017). Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal. International Journal of Microbiology, 2017, 1-5. doi: 10.1155/2017/2529085
2. Potenski, C., Gandhi, M., & Matthews, K. (2003). Exposure of Salmonella Enteritidis to chlorine or food preservatives increases susceptibility to antibiotics. FEMS Microbiology Letters, 220(2), 181-186. doi: 10.1016/s0378-1097(03)00099-5
3. KHATEEB, O., OSBORNE, D., & MULLA, Z. (2009). Gastrointestinal symptomatology as a predictor of severe outcomes of invasive group a streptococcal infections. Epidemiology And Infection, 138(4), 534-541. doi: 10.1017/s0950268809990938
4. KRONVALL, G., & RINGERTZ, S. (1991). Antibiotic disk diffusion testing revisited. Single strain regression analysis. APMIS, 99(1-6), 295-306. doi: 10.1111/j.1699-0463.1991.tb05153.x
5. Holman, D., & Gzyl, K. (2019). A meta-analysis of the bovine gastrointestinal tract microbiota. FEMS Microbiology Ecology, 95(6). doi: 10.1093/femsec/fiz072

Appendix

Dam Water Bacteria

Laboratory Report 1 - Bacteria Characteristics

Characteristics	Dam Water Red Colony	Dam Water Clear Colony
Cellular morphology	Gram reaction	Gram Negative	Gram Negative
Cellular morphology	Bacillus	Bacillus
Cell arrangement	Single	Single
Colony morphology	Gross morphology	Gross morphology
Colony height	Raised	Raised
Colony margins	Smooth	Smooth
Colony size	Medium	Large
Colony pigmentation	Red	Opaque

Colony Characteristics on Selective and Differential Media

Media	Dam Water Red Colony	Dam Water Clear Colony
Blood agar	y-haemolytic	y-haemolytic
MacConkey	Lactose fermenting Bacteria	Non lactose fermenting Bacteria
Mannitol salt agar	No growth	No growth
Brilliance chromogenic UTI agar	Blue	White

Biochemistry

Test	Dam Water Red Colony	Dam Water Clear Colony
Catalase test	Positive	Positive
Oxidase test	Negative	Positive

Identification

Identified Organism	Dam Water Red Colony	Dam Water Clear Colony
E. coli	Pseudomonas aeruginosa

Laboratory Report 3 - Antimicrobial Agents

Antibiotic	Dam Water Red Colony	Dam Water Clear Colony
Ampicillin	R	R
Gentamicin	26	21
Tetracycline	24	10
Chloramphenicol	14	23
Cephalothin	R	R
Clindamycin	R	R

Dish Cloth Bacteria

Laboratory Report 1 - Bacteria Characteristics

Characteristics	Dish Cloth Big Colony	Dish Cloth Small Colony
Cellular morphology	Gram reaction	Gram Negative	Gram Positive
Cellular morphology	Bacillus	Cocci
Cell arrangement	Single	Staph
Colony morphology	Gross morphology	Gross morphology
Colony height	Flat	Flat
Colony margins	Round	Round
Colony size	Medium	Large
Colony pigmentation	Opaque	Opaque

Colony Characteristics on Selective and Differential Media

Media	Dish Cloth Big Colony	Dish Cloth Small Colony
Blood agar	y-haemolytic	y-haemolytic
MacConkey	Lactose fermenting Bacteria	No growth
Mannitol salt agar	No growth	Mannitol Fermenting
Brilliance chromogenic UTI agar	Purple	White

Biochemistry

Test	Dish Cloth Big Colony	Dish Cloth Small Colony
Catalase test	Positive	Positive
Oxidase test	Negative	Negative

Identification

Identified Organism	Dish Cloth Big Colony	Dish Cloth Small Colony
E. coli	Staphylococcus aureus

Laboratory Report 3 - Antimicrobial Agents

Antibiotic	Dish Cloth Big Colony	Dish Cloth Small Colony
Ampicillin	20	20
Gentamicin	15	30
Tetracycline	17	25
Chloramphenicol	8	20
Cephalothin	10	30
Clindamycin	R	25